1. Field of the Invention
The present invention relates to techniques for controlling the expression of genes relating to biosynthesis of phenylpropanoid.
2. Description of the Background
With the advance of plant molecular biology in recent years, it has become possible to breed plants having useful characteristics, such as resistance to disease and insect damage or resistance to a herbicide, by using a sense gene or anti-sense gene. That is, the expression of a desired characteristic can be promoted or suppressed by linking, in a sense direction or anti-sense direction, a gene relating to the expression of the desired characteristic with a promoter permitting the expression in a plant to form a chimeric gene, and introducing the resulting chimeric gene to a plant as a vector. Based on such a technique, for example, a plant resistant to disease and insect damage to which an insecticidal BT toxin gene derived from Bacillus thuringensis has been introduced in a sense direction (D. A. Fischhoff et al., Bio/Technology, 232: 738-743 (1987)) and an excellent storable tomato to which a polygalacturonase gene relating to over-ripening of tomato fruit has been introduced in an anti-sense direction (C. J. Smith et al., Nature, 334: 724-727 (1988)) have been produced.
When such a technique is used, the expression of the desired characteristic is promoted in a plant to which a sense gene (a gene which expresses a desired characteristic and is fused to a promoter in a sense direction) has been introduced; on the other hand, the expression of the desired characteristic is inhibited in a plant to which an anti-sense gene (the same gene as the sense gene, which is fused to a promoter in an anti-sense direction) has been introduced. The expression of the desired characteristic is suppressed by the introduction of an anti-sense gene because in a plant cell, RNA synthesized from the anti-sense gene as a template is complementarily bound to mRNA derived from a gene of the plant itself relating to the expression of the desired characteristic to inhibit the subsequent synthesis of protein.
However, many genes of a plant form a multi-gene family and genes belonging to such a family show a high homology in a nucleotide sequence respectively. Even if the expression of such a gene belonging to a multi-gene family is controlled using the anti-sense method, the RNA of the anti-sense gene is inevitably bound at random to the mRNAs of many other genes belonging to the same family to control their expression, which makes it impossible to control the expression of only the desired gene, so that various characteristic suppression patterns is caused. Thus, the results are sometimes quite different from those as expected.
Also, the phenylpropanoid biosynthesis pathway is a complicatedly branched reaction system which exists specifically in plants and it relates to the biosynthesis of components of a cell wall (for example, lignin, suberin), pigments of a flower, antibacterial substances and the like. Phenylpropanoid derivatives available through such a biosynthesis pathway can also be used for UV protecting agents, insecticides or the like. If the expression of a gene relating to this phenylpropanoid biosynthesis pathway can be promoted or suppressed accurately, it becomes possible to control this biosynthesis pathway to produce a tree containing a lignin at a low content or carry out mass production of useful substances. In this case, however, it is difficult to control the expression of the gene by the anti-sense method owing to the above-described problem in homology between genes. For example, it is reported that a transformed plant to which a gene of phenylalanine ammonia lyase (PAL) or peroxidase (PRX) which is an enzyme acting in the phenylpropanoid biosynthesis pathway had been introduced in an anti-sense direction exhibited diversified controlling effects such as growth inhibition (M. M. Campbell and R. R. Sederoff, Plant Physiol., 110: 3-13 (1996)) and a change in a lignin content of tobacco to which a caffeic acid O-methyltransferase gene had been introduced in an anti-sense direction was not so large as expected (W. Ni et al., Transgen. Res., 3: 120-126 (1994)).